Multifunctional Forms of Polyoxazoline Copolymers and Drug Compositions Comprising the Same

ABSTRACT

The present disclosure provides copolymers of 2-substituted-2-oxazolines possessing two or three reactive functional groups which are also chemically orthogonal. The copolymers described may be random copolymers, block copolymers or a mixture of random and block copolymer configurations. Furthermore, the present disclosure provides novel methods for synthesizing the above polymers and for conjugating to molecules such as targeting, diagnostic and therapeutic agents.

The present application is a divisional of application Ser. No.12/744,472 (filed (May 24, 2010), which is the national stage ofInternational Application No. PCT/US2009/030762 (filed Jan. 12, 2009),which claims the benefit of U.S. Provisional Application No. 61/020,684(filed Jan. 11, 2008) and U.S. Provisional Application No. 61/029,337(filed Feb. 16, 2008).

FIELD OF THE DISCLOSURE

The present disclosure relates to multifunctional copolymers ofpolyoxazolines, methods of synthesis and intermediate compounds usefulin producing such polyoxazoline derivatives, and conjugates of thesepolyoxazolines with therapeutic, diagnostic and biological-targetingmolecules produced using such polyoxazoline derivatives.

BACKGROUND

Polymer-modified therapeutics have proven to be of great utility inmodern pharmaceutical science. Because of the success ofpolymer-modified therapeutics, it is of interest to expand the range ofpolymers suitable for such applications, especially to provide polymershaving properties not possessed by polymers of the prior art. A needexists for water-soluble, non-toxic polymers which can be used toprepare desired conjugates with target molecules. A need also exists forsuch polymers with multiple functionalities for use in preparing desiredconjugates with multiple molecules (such as, but not limited to,targeting moieties, therapeutic moieties and diagnostic moieties). Usingsuch polymers with multiple functionalities would allow the productionof conjugates containing one or more diagnostic and/or therapeuticmoiety or conjugates containing a mixture of distinct diagnostic and/ortargeting moieties. The present disclosure provides heterofunctionalpolyoxazoline compounds which provide ready coupling to a range ofmolecules, such as but not limited to, targeting, therapeutic and/ordiagnostic moieties.

DETAILED DESCRIPTION Definitions

As used herein, the term “POZ” or “POZ polymer” refers to a polymer of2-substituted-2-oxazoline containing a repeating unit having thestructure —[N(COR₂)CH₂CH₂]_(n)— in which R₂ is independently selectedfor each repeating unit from an unsubstituted or substituted alkyl,alkenyl, aralkyl or heterocyclylalkyl group and n is from 3-1000; in oneembodiment, the unsubstituted or substituted alkyl, alkenyl, aralkyl orheterocyclylalkyl groups comprise from 1-10 carbon atoms.

As used herein, the term “PMOZ” refers to POZ with the repeating unithaving the structure —[N(COCH₃)CH₂CH₂]_(n)—.

As used herein, the term “PEOZ” refers to POZ with the repeating unithaving the structure —[N(COCH₂CH₃)CH₂CH₂]_(n)—.

As used herein, the term M-POZ, M-PMOZ or M-PEOZ refers to the polymersabove in which the nitrogen of the initiating monomer unit is bound tomethyl.

As used herein, the term “POZ derivative” or “polyoxazoline derivative”refers to a structure comprising a POZ polymer, the POZ polymer havingat least one functional group capable of forming a linkage, directly orindirectly, with a chemical group on a target molecule, a linker or abranching moiety.

As used herein, the term “target molecule” refers to any molecule havingtherapeutic or diagnostic application or a targeting function, whereinthe target molecule is capable of reacting with a functional group on aPOZ polymer or a POZ derivative of the present disclosure, including,but not limited to, a drug, a diagnostic agent, a targeting molecule, anorganic small molecule, an oligonucleotide, a polypeptide, an antibodyor antibody fragment or a protein.

As used herein, the term “target molecule-POZ conjugate” refers to aconjugate of a POZ derivative of the present disclosure and at least onetarget molecule.

As used herein, the term “hydrolytically stable target molecule-POZconjugate” refers to a conjugate of a POZ derivative of the presentdisclosure and at least one target molecule, such that all the chemicallinkages in the conjugate are hydrolytically stable.

As used herein, the term “hydrolytically stable” refers to a linkagethat is stable in aqueous solutions under physiological conditions; inone embodiment, such linkages are stable for at least 12 hours, 24hours, 48 hours, 96 hours, 192 hours or greater; in an alternateembodiment such linkages are stable indefinitely.

As used herein, the term “hydrolytically unstable” refers to a linkagethat is not stable in aqueous solutions under physiological conditions.

As used herein, the term “physiological conditions” refers to an aqueoussolution having a pH from 6-8 and a temperature from 30-42 degreesCelsius.

As used herein, the term “functional group” refers to those groups thatwill react with a corresponding group, including those groups that willreact readily with electrophilic or nucleophilic groups, in contrast tothose groups that require strong catalysis or impractical reactionconditions in order to react.

As used herein, the term “link”, “linked” “linkage” or “linker” whenused with respect to a POZ derivative described herein, or componentsthereof, refers to groups or bonds that normally are formed as theresult of a chemical reaction and typically are covalent linkages.

As used herein, the term “protected” with respect to hydroxyl groups,amine groups, sulfhydryl groups and other reactive groups refers toforms of these functionalities which are protected from undesirablereaction with a protecting group known to those skilled in the art suchas those set forth in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, N.Y., (3rd Edition,1999) which can be added or removed using the procedures set forththerein. Examples of protected hydroxyl groups include, but are notlimited to, silyl ethers such as those obtained by reaction of ahydroxyl group with a reagent such as, but not limited to,t-butyldimethyl-chlorosilane, trimethylchlorosilane,triisopropylchlorosilane, triethylchlorosilane; substituted methyl andethyl ethers such as, but not limited to methoxymethyl ether,methythiomethyl ether, benzyloxymethyl ether, t-butoxymethyl ether,2-methoxyethoxymethyl ether, tetrahydropyranyl ethers, 1-ethoxyethylether, allyl ether, benzyl ether; esters such as, but not limited to,benzoylformate, formate, acetate, trichloroacetate, and trifluoracetate.Examples of protected amine groups include, but are not limited to,various alkyloxycarbonyls, amides such as, formamide, acetamide,trifluoroacetamide, and benzamide; imides, such as phthalimide, anddithiosuccinimide; and others. Examples of protected sulfhydryl groupsinclude, but are not limited to, thioethers such as S-benzyl thioether,and S-4-picolyl thioether; substituted S-methyl derivatives such ashemithio, dithio and aminothio acetals; and others.

As used herein, the team “alkyl”, whether used alone or as part of asubstituent or linking group, includes straight hydrocarbon groupscomprising from one to twenty carbon atoms. Thus the phrase includesstraight chain alkyl groups such as methyl, ethyl, propyl, butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and thelike. The phrase also includes branched chain isomers of straight chainalkyl groups, including but not limited to, the following which areprovided by way of example: —CH(CH₃)₂, —CH(CH₃)(CH₂CH₃), —CH(CH₂CH₃)₂,—C(CH₃)₃, —C(CH₂CH₃)₃, —CH₂ CH(CH₃)₂, —CH₂CH(CH₃)(CH₂CH₃),—CH₂CH(CH₂CH₃)₂, —CH₂C(CH₃)₃, —CH₂C(CH₂CH₃)₃, —CH(CH₃)CH(CH₃)(CH₂CH₃),—CH₂CH₂CH(CH₃)₂, —CH₂CH₂CH(CH₃)(CH₂CH₃), —CH₂CH₂CH(CH₂CH₃)₂,—CH₂CH₂C(CH₃)₃, —CH₂CH₂C(CH₂CH₃)₃, —CH(CH₃)CH₂CH(CH₃)₂,—CH(CH₃)CH(CH₃)CH(CH₃)CH(CH₃)₂, —CH(CH₂ CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), andothers. The phrase also includes cyclic alkyl groups such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl and such rings substituted with straight and branched chainalkyl groups as defined above. The phrase also includes polycyclic alkylgroups such as, but not limited to, adamantyl norbornyl, andbicyclo[2.2.2]octyl and such rings substituted with straight andbranched chain alkyl groups as defined above.

As used herein, the term “alkylene”, whether used alone or as part of asubstituent group, includes any group obtained by removing a hydrogenatom from an alkyl group; an alkylene group forms two bonds with othergroups.

As used herein, the term “alkenyl”, whether used alone or as part of asubstituent group, includes an alkyl group having at least one doublebond between any two adjacent carbon atoms.

As used herein, the term “alkynyl”, whether used alone or as part of asubstituent group, includes an alkyl group having at least one triplebond between any two adjacent carbon atoms.

As used herein, the term “unsubstituted alkyl”, “unsubstituted alkenyl”,and “unsubstituted alkynyl” refers to alkyl, alkenyl and alkynyl groupsthat do not contain heteroatoms.

The phrase “substituted alkyl”, “substituted alkenyl”, and “substitutedalkynyl” refers to alkyl, alkenyl and alkynyl groups as defined above inwhich one or more bonds to a carbon(s) or hydrogen(s) are replaced by abond to non-hydrogen or non-carbon atoms such as, but not limited to, ahalogen atom in halides such as F, Cl, Br, and I; and oxygen atom ingroups such as carbonyl, carboxyl, hydroxyl groups, alkoxy groups,aryloxy groups, and ester groups; a sulfur atom in groups such as thiolgroups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups,and sulfoxide groups; a nitrogen atom in groups such as amines, amides,alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines,N-oxides, imides, enamines imines, oximes, hydrazones, and nitriles; asilicon atom in groups such as in trialkylsilyl groups, dialkylarylsilylgroups, alkyldiarylsilyl groups, and triarylsilyl groups; and otherheteroatoms in various other groups. Other alkyl groups include those inwhich one or more bonds to a carbon or hydrogen atom is replaced by abond to an oxygen atom such that the substituted alkyl group contains ahydroxyl, alkoxy, aryloxy group, or heterocyclyloxy group. Still otheralkyl groups include alkyl groups that have an amine, alkylamine,dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine,heterocyclylamine, (alkyl)(heterocyclyl)-amine,(aryl)(heterocyclyl)amine, or diheterocyclylamine group.

As used herein, the term “unsubstituted aryl” refers to monocyclic orbicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in thering portion, such as, but not limited to, phenyl, naphthyl,anthracenyl, biphenyl and diphenyl groups, that do not containheteroatoms. Although the phrase “unsubstituted aryl” includes groupscontaining condensed rings such as naphthalene, it does not include arylgroups that have other groups such as alkyl or halo groups bonded to oneof the ring members, as aryl groups such as tolyl are considered hereinto be substituted aryl groups as described below. Unsubstituted arylgroups may be bonded to one or more carbon atom(s), oxygen atom(s),nitrogen atom(s), and/or sulfur atom(s) in the parent compound, however.

As used herein, the term “substituted aryl group” has the same meaningwith respect to unsubstituted aryl groups that substituted alkyl groupshad with respect to unsubstituted alkyl groups. However, a substitutedaryl group also includes aryl groups in which one of the aromaticcarbons is bonded to one of the non-carbon or non-hydrogen atoms, suchas, but not limited to, those atoms described above with respect to asubstituted alkyl, and also includes aryl groups in which one or morearomatic carbons of the aryl group is bonded to a substituted and/orunsubstituted alkyl, alkenyl, or alkynyl group as defined herein. Thisincludes bonding arrangements in which two carbon atoms of an aryl groupare bonded to two atoms of an alkyl, alkenyl, or alkynyl group to definea fused ring system (e.g. dihydronaphthyl or tetrahydronaphthyl). Thus,the phrase “substituted aryl” includes, but is not limited to tolyl, andhydroxyphenyl among others.

As used herein, the term “unsubstituted aralkyl” refers to unsubstitutedor substituted alkyl, alkenyl or alkynyl groups as defined above inwhich a hydrogen or carbon bond of the unsubstituted or substitutedalkyl, alkenyl or alkynyl group is replaced with a bond to an aryl groupas defined above. For example, methyl (CH₃) is an unsubstituted alkylgroup. If a hydrogen atom of the methyl group is replaced by a bond to aphenyl group, such as if the carbon of the methyl were bonded to acarbon of benzene, then the compound is an unsubstituted aralkyl group(i.e., a benzyl group).

As used herein, the term “substituted aralkyl” has the same meaning withrespect to unsubstituted aralkyl groups that substituted aryl groups hadwith respect to unsubstituted aryl groups. However, a substitutedaralkyl group also includes groups in which a carbon or hydrogen bond ofthe alkyl part of the group is replaced by a bond to a non-carbon or anon-hydrogen atom.

As used herein, the term “unsubstituted heterocyclyl” refers to botharomatic and nonaromatic ring compounds including monocyclic, bicyclic,and polycyclic ring compounds such as, but not limited to, quinuclidyl,containing 3 or more ring members of which one or more is a heteroatomsuch as, but not limited to, N, O, and S. Although the phrase“unsubstituted heterocyclyl” includes condensed heterocyclic rings suchas benzimidazolyl, it does not include heterocyclyl groups that haveother groups such as alkyl or halo groups bonded to one of the ringmembers, as compounds such as 2-methylbenzimidazolyl are “substitutedheterocyclyl” groups as defined below. Examples of heterocyclyl groupsinclude, but are not limited to: unsaturated 3 to 8 membered ringscontaining 1 to 4 nitrogen atoms such as, but not limited to pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl,pyrazinyl, pyridazinyl, triazolyl, tetrazolyl; saturated 3 to 8 memberedrings containing 1 to 4 nitrogen atoms such as, but not limited to,pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensedunsaturated heterocyclic groups containing 1 to 4 nitrogen atoms suchas, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl;unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1to 3 nitrogen atoms such as, but not limited to, oxazolyl, isoxazolyl,oxadiazolyl; saturated 3 to 8 membered rings containing 1 to 2 oxygenatoms and 1 to 3 nitrogen atoms such as, but not limited to,morpholinyl; unsaturated condensed heterocyclic groups containing 1 to 2oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl,benzoxadiazolyl, benzoxazinyl (e.g. 2H-1,4-benzoxazinyl etc.);unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atoms and 1to 3 nitrogen atoms such as, but not limited to, thiazolyl,isothiazolyl, thiadiazolyl (e.g. 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); saturated 3 to 8 memberedrings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as,but not limited to, thiazolodinyl; saturated and unsaturated 3 to 8membered rings containing 1 to 2 sulfur atoms such as, but not limitedto, thienyl, dihydrodithiinyl, dihydrodithionyl, tetrahydrothiophene,tetrahydrothiopyran; unsaturated condensed heterocyclic rings containing1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limitedto, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g.2H-1,4-benzothiazinyl, etc.), dihydrobenzothiazinyl (e.g.2H-3,4-dihydrobenzothiazinyl, etc.), unsaturated 3 to 8 membered ringscontaining oxygen atoms such as, but not limited to furyl; unsaturatedcondensed heterocyclic rings containing 1 to 2 oxygen atoms such asbenzodioxolyl (e.g. 1,3-benzodioxoyl, etc.); unsaturated 3 to 8 memberedrings containing an oxygen atom and 1 to 2 sulfur atoms such as, but notlimited to, dihydrooxathiinyl; saturated 3 to 8 membered ringscontaining 1 to 2 oxygen atoms and 1 to 2 sulfur atoms such as1,4-oxathiane; unsaturated condensed rings containing 1 to 2 sulfuratoms such as benzothienyl, benzodithiinyl; and unsaturated condensedheterocyclic rings containing an oxygen atom and 1 to 2 oxygen atomssuch as benzoxathiinyl. Heterocyclyl group also include those describedabove in which one or more S atoms in the ring is double-bonded to oneor two oxygen atoms (sulfoxides and sulfones). For example, heterocyclylgroups include tetrahydrothiophene, tetrahydrothiophene oxide, andtetrahydrothiophene 1,1-dioxide. Preferred heterocyclyl groups contain 5or 6 ring members. More preferred heterocyclyl groups includemorpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole,1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine,thiomorpholine in which the S atom of the thiomorpholine is bonded toone or more O atoms, pyrrole, homopiperazine, oxazolidin-2-one,pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan, andtetrahydrofuran.

As used herein, the term “substituted heterocyclyl” has the same meaningwith respect to unsubstituted heterocyclyl groups that substituted alkylgroups had with respect to unsubstituted alkyl groups. However, asubstituted heterocyclyl group also includes heterocyclyl groups inwhich one of the carbons is bonded to one of the non-carbon ornon-hydrogen atom, such as, but not limited to, those atoms describedabove with respect to a substituted alky and substituted aryl groups andalso includes heterocyclyl groups in which one or more carbons of theheterocyclyl group is bonded to a substituted and/or unsubstitutedalkyl, alkenyl, alkynyl or aryl group as defined herein. This includesbonding arrangements in which two carbon atoms of an heterocyclyl groupare bonded to two atoms of an alkyl, alkenyl, or alkynyl group to definea fused ring system. Examples, include, but are not limited to,2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl,1-methyl piperazinyl, and 2-chloropyridyl among others.

As used herein, the term “unsubstituted heterocyclylalkyl” refers tounsubstituted or substituted alkyl, alkenyl or alkynyl groups as definedabove in which a hydrogen or carbon bond of the unsubstituted orsubstituted alkyl, alkenyl or alkynyl group is replaced with a bond to aheterocyclyl group as defined above. For example, methyl (CH₃) is anunsubstituted alkyl group. If a hydrogen atom of the methyl group isreplaced by a bond to a heterocyclyl group, such as if the carbon of themethyl were bonded to carbon 2 of pyridine (one of the carbons bonded tothe N of the pyridine) or carbons 3 or 4 of the pyridine, then thecompound is an unsubstituted heterocyclylalkyl group.

As used herein, the term “substituted heterocyclylalkyl” has the samemeaning with respect to unsubstituted heterocyclylalkyl groups thatsubstituted aryl groups had with respect to unsubstituted aryl groups.However, a substituted heterocyclylalkyl group also includes groups inwhich a non-hydrogen atom is bonded to a heteroatom in the heterocyclylgroup of the heterocyclylalkyl group such as, but not limited to, anitrogen atom in the piperidine ring of a piperidinylalkyl group.

General Description

Polyoxazolines (POZ) are polymers prepared from2-substituted-2-oxazoline monomers. These polymers are water soluble andhave been reported to be nontoxic in mammalian model systems. POZ isgenerally prepared by reaction of the appropriate stoichiometric amountof 2-alkyl-2-oxazoline with an electrophilic initiator, such as methyltriflate (CH₃—OSO₂—CF₃) or a strong acid such as triflic acid orp-toluenesulfonic acid, followed by termination with a nucleophile suchas, but not limited to, hydroxide, a thiol or an amine. The polymerproduced is conveniently described in shorthand with the initiatinggroup designated by the leftmost group and the terminating groupdesignated by the rightmost group, with the 2-alkyl-2-oxazolinecomponent in the middle. Therefore, when this shorthand description isused in the current specification, it is intended that the left side ofthe designation presents the “initiator end” and the right side of thedesignation presents the “termination end”, unless designated otherwise.

For example, when the 2-alkyl-2-oxazoline is 2-methyl-2-oxazoline,methyl triflate is used as the initiator and hydroxide is used as theterminator, the following POZ is produced:

CH₃—[N(COCH₃)CH₂CH₂]—OH

The polymer above is conveniently described in shorthand notation asM-PMOZ-OH, in which the methyl initiator is designated by the leftmostM, PMOZ represents polymethyloxazoline with the methyl of the repeatingunit designated by the M of PMOZ, and the terminating hydroxyl isdesignated by the —OH.

Another commonly used monomer is 2-ethyl-2-oxazoline, which with methyltriflate initiation and hydroxide termination would provide thefollowing POZ polymer:

CH₃—[N(COCH₂CH₃)CH₂CH₂]_(n)—OH

The polymer above is conveniently described in shorthand notation asM-PEOZ-OH, in which the methyl initiator is designated by the leftmostM, PEOZ represents polymethyloxazoline with the ethyl of the repeatingunit designated by the E of PEOZ, and the terminating hydroxyl isdesignated by the —OH.

The degree of polymerization, n, for well characterized polymers canrange from approximately 3 to about 1000.

The polymerization process is referred to as a living, cationicpolymerization since initiation with an electrophile produces anoxazolinium cation that then reacts in a chain reaction with additionalmonomer units to produce a growing, “living” cation.

One can predict the products of termination by assuming that the livingcation can be represented in the following non-cyclic form (forpolymerization of 2-methyl-2-oxazoline initiated with methyl triflate),although in reality the cyclic form is certainly the most important:

CH₃—[N(COCH₃)CH₂CH₂]_(n)—N(COCH₃)CH₂CH₂ ⁺

In the current discussion we will represent this cation as M-PMOZ⁺. Asnoted above, this POZ cation can be “terminated” by reacting withnucleophiles such as hydroxide, thiols or amines,

Oxazoline polymerization can also be initiated with functionalelectrophiles. For example the electrophilic initiator ethyl3-bromopropionate has been used to initiate 2-ethyl-2-oxazolinepolymerization. Tciinination with hydroxide gives the following polymer:

HO₂C—CH₂CH₂—[N(COCH₂CH₃)CH₂CH₂]_(n), —OH

Yet another route to preparing polyoxazolines with functional groups isto copolymerize a monomer such as 2-ethyl-2-oxazoline with an oxazolinemonomer having a functional group in the 2-position (F. C. Gaertner, R.Luxenhofer, B. Blechert, R. Jordan and M. Essler, J. Controlled Release,2007, 119, 291-300). For example, Jordan and colleagues have preparedoxazolines with acetylenes and protected aldehydes, carboxylic acids andamines in the 2-position. Copolymerization of these functional monomerswith 2-ethyl-2-oxazoline gives random copolymers with multiple pendentor side-chain functional groups. For example, initiation with methyltriflate of polymerization of 2-ethyl-2-oxazoline and2-pentynyl-2-oxazoline, followed by termination with piperazine(NHC₄H₈NH) gives the following random copolymer:

CH₃—{[N(COCH₂CH₃)CH₂CH₂]_(n)—[N(COCH₂CH₂CH₂—CCH)CH₂CH₂]_(m)}_(ran)—NC₄H₈NH

The subscript “ran” indicates that the polymer is a random copolymer.Values of n are typically around 20-30 while m is around 2-5.

These copolymers with pendent functional groups and a terminalfunctional group are useful in that the pendent and terminal functionalgroups can be “chemically orthogonal” functional groups. Chemicallyorthogonal functional groups are those functional groups that will notreact with each other but will react selectively with other functionalgroups. For example, the molecule above has two functional groups, aterminal secondary amine and pendent acetylenes. The acetylene will notreact with the amine but will, for example, react with an azide group(—N₃). Similarly, the amine will not react with acetylene or azide butwill react with, for example, an isothiocyanate group (—NCS). Jordan hasused this copolymer to couple an azide-functionalized RGD peptide to theacetylene group, and an isothiocyanate-functionalized metal chelator tothe amine The RGD peptide is known to target tumors, and a diagnostic ortherapeutic radionuclide can bind to the chelating group. The finalconjugate can be used to image or treat tumors (R. Luxenhofer, M.Lopez-Garcia, A. Frannk, H. Kessler and R. Jordan, Proceedings of theAmerican Chemical Society, PMSE Prepr. 2006, 95, 283-284).

One problem hindering use of the above piperazine- orpiperidine-terminated polyoxazolines is that they are difficult topurify. This difficulty arises because contaminating water presentduring termination leads to nucleophilic attack by water and consequentformation of secondary amine impurity (O. Nuyken, G. Maier, A. Gross,Macromol. Chem. Phys. 197, 83-95, 1996). Since the products frompiperazine and piperidine termination always contain a tertiary amine,ion-exchange chromatography cannot be used to remove the contaminatingsecondary amine

Still another limitation of the pendent polyoxazolines illustrated aboveis that these compounds possess a single terminal functional group.Consequently, this structural configuration limits the number of drug ortargeting moieties that can be attached to the terminus, whereaseffective use of such compounds for therapeutic diagnostic and targetingapplications may require multiple loading of these moieties. Thepolymers of the current disclosure avoid this limitation by providingfor multiple copies of each of two chemically reactive and orthogonalfunctional groups.

Novel Heterofunctional Polyoxazoline Derivatives

The present disclosure avoids the limitations of the prior art byproviding heterofunctional polyoxazoline derivatives of two2-substituted-2-oxazolines comprising at least two functional groupswhich are chemically reactive and chemically orthogonal to one another.The heterofunctional polyoxazoline derivatives may contain additionalfunctional groups as well. In certain embodiments, all functional groupsare chemically orthogonal to one another, while in other embodiments,the additional functional groups may be chemically orthogonal to atleast one other functional group present on the heterofunctionalpolyoxazoline derivative.

Single-Arm Heterofunctional Polyoxazoline Derivatives

In one embodiment, the heterofunctional polyoxazoline derivative isrepresented by the general structure:

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COY)CH₂—CH₂)]_(m)}_(a)—Z

-   wherein:-   R₁ is an initiating group;-   X is a pendent moiety containing a first functional group;-   Y is a pendent moiety containing a second functional group;-   Z is a terminating nucleophile; in certain embodiments Z is inert    (i.e., does not contain a functional group); in other embodiments, Z    contains a third functional group;-   a is ran which indicates a random copolymer or block which indicates    a block copolymer; and-   o and m are each an integer independently selected from 1-50.

Exemplary initiating groups include, but are not limited to, hydrogen,alkyl, substituted alkyl, aralkyl, or substituted aralkyl groups. In aparticular embodiment, the initiating group is a methyl group. The R₁group is selected to lack a functional group. Additional exemplaryinitiating groups are disclosed in PCT Application No.PCT/US2008/078159, which is hereby incorporated by reference for suchteaching.

As shown by the general structure above, the heterofunctionalpolyoxazoline derivative comprises at least two functional groups. Thefirst and second functional groups are present on the pendent moieties Xand Y, respectively. In certain embodiment, a third functional group ispresent on the Z group; however, the presence of the third functionalgroup is optional and in some embodiments Z may be inert (i.e., lackingthe third functional group).

X and Y are pendent moieties bearing first and second functional groups,respectively. In certain embodiments, X and Y may contain a linkingportion that links the first and second functional groups to thepolyoxazoline derivative. Exemplary linking portions include alkylenegroups. In certain cases, the alkylene group is a C₁-C₁₅ alkylene group.The linking portions of X and Y may be the same or may be different. Forexample, both X and Y may contain a C₅ alkylene group as the linkingportion or X may contain a C₅ alkylene group as the linking portion andY may contain a C₈ alkylene group as the linking portion.

The first and second functional groups are chemically orthogonal to oneanother. The first and second functional groups include, but are notlimited to, alkyne, amine, oxyamine, aldehyde, ketone, acetal, ketal,maleimide, ester, carboxylic acid, activated carboxylic acid (such as,but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazineylactive ester), an active carbonate, a chloroformate, alcohol, azide,vinyl sulfone, or orthopyridyl disulfide (OPSS), provided that theselection is made for X and Y so that the first and second functionalgroups are chemically orthogonal to one another.

As discussed above, Z may contain a third functional group or be inert.In those embodiments where Z contains a third functional group, thethird functional group may be chemically orthogonal to one or both ofthe first and second functional groups. Exemplary third functionalgroups include, but are not limited to, alkyne, amine, oxyamine,aldehyde, ketone, acetal, ketal, maleimide, ester, carboxylic acid,activated carboxylic acid (such as, but not limited to,N-hydroxysuccinimidyl (NHS) and 1-benzotriazineyl active ester), anactive carbonate, a chloroformate, alcohol, azide, vinyl sulfone, ororthopyridyl disulfide (OPSS).

In a particular embodiment, Z contains the third functional group and isrepresented by the structure —S—U—W, wherein S is a sulphur atom, U is alinking group and W is the third functional group. In this embodiment,representative U groups include alkylene groups. In a particularembodiment, U is —(CH₂)_(p)— where p is an integer selected from 1 to10. In a particular embodiment, W may be a carboxylic acid, a protectedcarboxylic acid, an active ester, an amine or a protected amineFurthermore, W may be selected from the groups described above for Z. Asdiscussed herein, the third functional group may be chemicallyorthogonal to one or both of the first functional groups. Embodiments ofthe foregoing include but are not limited to:

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COY)CH₂—CH₂)]_(m)}_(a)—S—(CH₂)_(p)—NH₂ and

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COY)CH₂—CH₂)]_(m)}_(a)—S—(CH₂)_(p)—CO₂H.

In an alternate embodiment, Z lacks the third functional group and isrepresented by the structure —S-T-V, wherein S is a sulphur atom, T is alinking group and V is an inert group. In this embodiment,representative T groups include alkylene groups. In a particularembodiment, T is —(CH₂)_(p)— where p is an integer selected from 1 to10. V may be any inert group. In a particular embodiment, V is —CO₂CH₃or —C₆H₅.

Embodiments of the foregoing include but are not limited to:

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COY)CH₂—CH₂)]_(m)}_(a)—S—(CH₂)_(p)—C₆H₅ and

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COY)CH₂—CH₂)]_(m)}_(a)—S—(CH₂)_(p)—CO₂CH₃.

In an alternate embodiment, the heterofunctional polyoxazolinederivative is represented by the general structure below.

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂—CH₂)]_(m)}_(a)—Z

-   wherein:-   R₁, X, Y, Z, o, m and a are as defined above;-   R₂ lacks a functional group and is independently selected for each    repeating unit from an unsubstituted or substituted alkyl, an    unsubstituted or substituted alkenyl, an unsubstituted or    substituted aralkyl or an unsubstituted or substituted    heterocyclylalkyl group.

In this embodiment, an additional 2-alkyl-2-oxaxoline is introduced as athird co-monomer. This third 2-alkyl-2-oxaxoline co-monomer lacks afunctional group and provides a chemically unreactive spacer between thefirst and second functional groups present on X and Y, respectively.Such a configuration prevents the first and second functional groupsfrom being sterically hindered.

In certain embodiment, o and m are not zero and the heterofunctionalpolyoxazoline derivative contains first and second functional groups onX and Y respectively. The first and second functional groups may beselected from the groups described above. As above, the first and secondfunctional groups are chemically orthogonal to one another.

The Z group, as discussed above may contain a third functional group orbe inert. In those embodiments where Z contains a third functionalgroup, the third functional group may be chemically orthogonal to one orboth of the first and second functional groups. When Z contains thethird functional group, the third functional group may be selected fromthe groups described above.

In a particular embodiment, Z contains the third functional group and isrepresented by the structure —S—U—W, wherein S is a sulphur atom, U is alinking group and W is the third functional group. In this embodiment,representative U groups include alkylene groups. In a particularembodiment, U is —(CH₂)_(p)— where p is an integer selected from 1 to10. In a particular embodiment, W may be a carboxylic acid, a protectedcarboxylic acid, an active ester, an amine or a protected amine.Furthermore, W may be selected from the groups described above for Z. Asdiscussed herein, the third functional group may be chemicallyorthogonal to one or both of the first functional groups.

Embodiments of the foregoing include but are not limited to:

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂)]_(m)}_(a)—S—(CH₂)_(p)—NH₂andR₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂)]_(m)}_(a)—S—(CH₂)_(p)—CO₂H.

In an alternate embodiment, Z lacks the third functional group and isrepresented by the structure —S-T-V, wherein S is a sulphur atom, T is alinking group and V is an inert group. In this embodiment,representative T groups include alkylene groups. In a particularembodiment, T is —(CH₂)_(p)— where p is an integer selected from 1 to10. V may be any inert group. In a particular embodiment, V is —CO₂CH₃or —C₆H₅.

Embodiments of the foregoing include but are not limited to:

R₁{[N(COX)CH₂CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂)]_(n)}_(a)—S—(CH₂)_(p)—C₆H₅andR₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂)]_(n)}_(a)—S—(CH₂)_(p)—CO₂CH₃.

In still another alternate embodiment, the heterofunctionalpolyoxazoline derivatives are defined by the general structure:

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)}_(a)—S—U—W

-   wherein:-   R₁, X, R₂, o, n and a are as defined above;-   S is a sulphur atom;-   U is a linking group; and-   W is the third functional group.

In this embodiment, representative U groups include alkylene groups. Ina particular embodiment, U is —(CH₂)_(p)— where p is an integer selectedfrom 1 to 10. In this embodiment, the second functional group islacking. In these embodiments, Z contains a third functional group andthe third functional group is chemically orthogonal to the firstfunctional group on X. The first functional group may be selected fromthe groups described above. The third functional group W may be selectedfrom the groups described above for X. In a particular embodiment, W maybe a carboxylic acid, a protected carboxylic acid, an active ester, anamine or a protected amine.

Embodiments of the foregoing include but are not limited to:

R₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)}_(a)—S—(CH₂)_(p)—CO₂ and

R₁{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)}_(a)—S—(CH₂)_(p)—NH₂.

These heterofunctional polyoxazoline derivatives described herein may beprepared by terminating the POZ cation with a mercapto-ester (such as—S—CH₂CH₂—CO₂CH₃) or mercapto-protected amine (such as—S—CH₂CH₂—NH-tBoc). These heterofunctional polyoxazoline derivativesprovide for effective, large-scale purification by ion-exchangechromatography (to remove secondary amines), and they provide chemicallyorthogonal functional groups X and W (—CO₂H or —NH₂) for attachment ofone or more target molecules, such as targeting, diagnostic ortherapeutic moieties.

Multi-arm Heterofunctional Polyoxazoline Derivatives

The present disclosure also provides for multi-armed heterofunctionalpolyoxazoline derivatives. The multi-armed heterofunctionalpolyoxazoline derivatives may contain from 2 to 8 polyoxazoline chains.In a particular embodiment, the multi-armed heterofunctionalpolyoxazoline derivatives contain 2 or 4 polyoxazoline chains. Themulti-armed heterofunctional polyoxazoline derivatives may berepresented by the general formula:

{R₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂)]_(m)}_(a)—K_(k)—}_(d)—R-Q_(q)-Z

-   wherein:-   R₁ is an initiating group;-   R₂ lacks a functional group and is independently selected for each    polyoxazoline chain from a substituted or unsubstituted alkyl, a    substituted or unsubstituted alkenyl, a substituted or unsubstituted    aralkyl or a substituted or unsubstituted heterocyclylalkyl group;-   X is a pendent moiety bearing a first functional group;-   Y is a pendent moiety bearing a second functional group;-   K is a linking moiety linking each polyoxazoline chain to a    branching moiety R;-   Q is a linking moiety linking the branching moiety R to Z;-   R is a branching moiety capable of forming linkages with Z, either    directly or through linking group Q, and with each polyoxazoline    chain, either directly or through the linking group K;-   Z is a moiety containing a third functional group or an inert group;

A is independently selected for each polyoxazoline chain from ran whichindicates a random copolymer or block which indicates a block copolymer;

-   d is an integer selected from 2-8;-   k is an integer independently selected for each polyoxazoline chain    from one or zero;-   q is an integer selected from one or zero;-   o is an integer independently selected for each polyoxazoline chain    from 1-50;-   m is an integer independently selected for each polyoxazoline chain    from 0-50;-   n is an integer independently selected for each polyoxazoline chain    from 0-1000; and-   wherein at least two of the first, second and third functional    groups may be chemically orthogonal to one another.

In the general structure above, each POZ chain may be the same or may bedifferent. Furthermore, the pendent moieties X and Y may be the same ormay be different for each POZ chain.

Exemplary R₁ groups include, but not limited to, hydrogen, alkyl,substituted alkyl, aralkyl, or substituted aralkyl groups. In aparticular embodiment, the initiating group is a methyl group. The R₁group is selected to lack a functional group. Additional exemplaryinitiating groups are disclosed in PCT Application No.PCT/US2008/078159, which is hereby incorporated by reference for suchteaching.

As shown by the general structure above, the heterofunctionalpolyoxazoline derivative comprises at least two functional groups. Thefirst and second functional groups are present on the pendent moieties Xand Y, respectively. In certain embodiment, a third functional group ispresent on the Z group; however, the presence of the third functionalgroup is optional and in some embodiments Z may be inert (i.e., lackingthe third functional group).

X and Y are pendent moieties bearing first and second functional groups,respectively. In certain embodiments, X and Y may contain a linkingportion that links the first and second functional groups to thepolyoxazoline derivative. Exemplary linking portions include alkylenegroups. In certain cases, the alkylene group is a C₁-C₁₅ alkylene group.The linking portions of X and Y may be the same or may be different. Forexample, both X and Y may contain a C₅ alkylene group as the linkingportion or X may contain a C₅ alkylene group as the linking portion andY may contain a C₈ alkylene group as the linking portion.

Z may contain a third functional group or be inert. In those embodimentswhere Z contains a third functional group, the third functional groupmay be chemically orthogonal to one or both of the first and secondfunctional groups.

The first, second and third functional groups include, but are notlimited to, alkyne, amine, oxyamine, aldehyde, ketone, acetal, ketal,maleimide, ester, carboxylic acid, activated carboxylic acid (such as,but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazineylactive ester), an active carbonate, a chloroformate, alcohol, azide,vinyl sulfone, or orthopyridyl disulfide (OPSS), provided that theselection is made so that at least two of the first, second and thirdfunctional groups are chemically orthogonal to one another.

Q is optional and can be any group capable of forming linkages with boththe R and Z and will be different depending on the chemistry of the Rand Z. Representative Q groups include, but are not limited to,substituted and unsubstituted alkylene groups. In a specific embodiment,Q is —(CH₂)_(p)—, where p is independently selected from 1-10.

K is optional and can be any group capable of forming linkages with boththe POZ chain and R and will be different depending on the chemistry ofthe POZ chain and R. Representative K groups include substituted andunsubstituted alkyl, alkenyl or alkynyl groups. In a specificembodiment, K is —(CH₂)_(p)O—, —(CH₂)_(p)—CO—, —S—(CH₂)_(p)CONH—,—S—(CH₂)_(p)CO, —(CH₂)_(p)—NHCSO—, —(CH₂)_(p)—NHCO₂—, —NH—(CH₂)_(p), or—NHCO₂—, where p is an integer from 0-10. K may be the same or differentfor each POZ chain.

R is a branching moiety capable of forming linkages with both POZ chainsand Z, either directly or through linking groups K and Q, respectively.R may be selected from a nitrogen, an aryl group, or —CR₃—, where R₃ ishydrogen or a substituted or unsubstituted alkyl, a substituted orunsubstituted alkenyl, or a substituted or unsubstituted aralkyl group.In a specific embodiment, R is —NH—CH—(CH₂)₃—CH₂—NH— or—(CH₂)₄—CH—CO—NH—(CH₂)₄—CH—NH—CO—CH—(CH₂)₄—.

In the general structure above, at least two of the first, second andthird functional groups must be present. Therefore, when m or n is 0 foreach POZ chain, Z cannot be inert.

Several embodiments of the multi-aimed heterofunctional polyoxazolinederivative falling under the general structure above are presentedbelow.

Example 1

In this example, d is 2, k is 1, K is —S—(CH₂)_(p)—CO—, where p is aninteger from 1-10, for each polyoxazoline chain, q is 0, R isNH—CH—(CH₂)₃—CH₂—NH and the first and second functional groups arechemically orthogonal to one another; Z may be chemically orthogonal toone or both of the first and second functional groups.

Example 2

In this example, d is 2, k is 1, K is —S—(CH₂)_(p)—CO—, where p is aninteger from 1-10, for each polyoxazoline chain, q is 0, R isNH—CH—(CH₂)₃—CH₂—NH, m is 0, Z is CO₂H and the first and thirdfunctional groups may be chemically orthogonal to one another.

Example 3

In this example, d is 4, k is 1 and K is —S—(CH₂)₂—CO—NH for eachpolyoxazoline chain, q and m are 0, R is—(CH₂)₄—CH—CO—NH—CO—(CH₂)₄—CH—NH—CO—CH—(CH₂)₄— and the first and thirdfunctional groups may be chemically orthogonal to one another.

Example 4

In this example, d, k, K, q, m and R are as in Example 3 and Z is CO₂Hand the first and third functional groups may be chemically orthogonalto one another.

In all of the above embodiments, the polyoxazoline polymers contained inthe heterofunctional polyoxazoline derivatives may be random or blockcopolymers. As used herein, a block copolymer includes those copolymersthat have block configurations separated by a random copolymer sequence.Such random and block copolymers may be produced by controlling theintroduction of various intermediates during the synthesis process asdescribed below.

Furthermore, in all the embodiments discussed above, one or more of thefunctional groups may be charged species. For example, one or more ofthe functional groups may be a carboxylic acid having a negative chargeor an amine having a positive charge. As such, the functional groups mayform ionic linkages with one or more target molecules.

Methods of Synthesis

In one embodiment, the polymers of the present disclosure are preparedby co-polymerization of appropriate 2-substututed-2-oxazolinescontaining functional groups. For example, preparation of awater-soluble co-polymer bearing alkyne groups and acetal groups, inaddition to a simple 2-alkyl-2-oxazoline can be synthesized using thefollowing oxazoline monomers:

The polymerization is initiated by an electrophile such as, but notlimited to, methyl triflate, methyl tosylate, p-toluenesulfonic acid, ortriflic acid. The co-polymerization can be terminated by a nucleophilicreagent as discussed herein. If terminal functionality is desired, afunctionalized terminating agent, such as but not limited to, methylthiolacetate can be used. For a non-reactive termination terminus, anucleophile such as an alkyl mercaptan can be used. Similarly aterminating hydroxyl group is also unreactive to many reagents and cantherefore be useful in this application. Preferred solvents for thepolymerization are chlorobenzene or acetonitrile. The preferredtemperature range is from about 40° C. to about 120° C. The timerequired for the polymerization is dependent on the temperature, thedesired molecular weight, and the solvent and can range from about 1 hto about 100 h. In certain embodiments, it is desirable to limit thepolymerization reaction to the time required to substantially completethe polymerization reaction. In one embodiment, the progress of thepolymerization reaction is monitored using MALDI and/or GPC.

The polymerization can be conducted in several ways. In one embodiment,a mixture of the appropriate oxazoline components can be reacted withthe initiator in a preferred solvent with stirring. This reaction yieldsa random copolymer when the oxazoline components are equally reactivewith one another or a block copolymer when one or more of the oxazolinecomponents are less reactive towards one another. In an alternateembodiment, the polymer may also be synthesized in blocks by initiatingpolymerization with an appropriate initiator in a preferred solventusing only one oxazoline component. The reaction may be monitored withMALDI or GPC to determine when the reaction is substantially complete.When polymerization of the first block is complete, a second oxazolinecomponent is added to reinitiate the polymerization with the incipientliving cation at the terminus of the polymer chain; monitoring of thepolymerization reaction may be carried out as described. The solvent maybe the same as in the first polymerization reaction or different; thereaction temperature and other variables may also be adjusted asdesired. Upon completion of the polymerization of the second block, athird oxazoline component is added to reinitiate the polymerization withthe living cation at the terminus of the polymer chain; monitoring ofthe polymerization reaction may be carried out as described. The solventmay be the same as in the first or second polymerization reactions ordifferent; the reaction temperature and other variables may also beadjusted as desired. When the third polymerization step is completed,the polymerization may be terminated by the addition of a terminatingagent. The oxazoline components may contain functional groups, or maylack a functional group. The sequential polymerizations can be done inany order. In another embodiment, random copolymerization of two of theoxazoline monomers, followed by continued polymerization of a thirdblock can also be done to produce a polymer having both random and blockconfigurations. In one embodiment, one of the oxazoline monomers lacks afunctional group.

Once the desired polymerization process is completed, the polymer isprecipitated, such as in ethyl ether, several times and dried undervacuum. The polymer may be further characterized by standard techniquessuch as but not limited to MALDI, NMR, and GPC.

Work with polyethylene glycol has shown that it is frequently necessaryin modification of target molecules to utilize polymers of molecularweights (MWs) of 20,000 Da or higher and molecular weight distributions,or polydispersities (PDs), of less than 1.1. There has been a great dealof work showing that MWs and PDs in the above range cannot be achievedfor POZ chains with conventional techniques. As is known in the art PDvalues will vary with MW; in general, as the molecular weight increasesthe PD value also increases. It is generally seen that as the molecularweight of growing POZ chains reaches approximately 5,000 Da, thepolydispersity increases appreciably. Side reactions, including, but notlimited to, chain transfer, begin to grow in importance. The prior arttechniques described above when used to generate POZ chains of high MWproduce POZ derivatives with unacceptable PD values. The polyoxazolinederivatives of the present disclosure may be produced usingpolyoxazoline chains that are manufactured using novel methods thatresult in polyoxazoline derivatives with low PD values and a decreasedamount of impurities produced by unwanted side reactions, such as, butnot limited to, chain transfer. In one embodiment, the polyoxazolinechains are manufactured to minimize unwanted side reactions, such as,but not limited to, chain transfer, allowing the production of POZderivatives of increased purity with low PD values. Therefore, the POZderivatives of the present disclosure may be produced with increasedpurity and with low PD values suitable for use in pharmaceuticalapplications. Such methods are described in PCT Application No.PCT/US2008/078159, which is hereby incorproated by reference for suchteaching

Use of the Heterofunctional Polyoxazoline Derivatives

The heterofunctional polyoxazoline derivatives of the present disclosurepossess two or more chemically orthogonal functional groups and thusallow for attachment of two or more different target molecules to thepolymer.

The novel heterofunctional polyoxazoline derivatives prepared asdescribed above are intended for formation of conjugates with varioustarget molecules, such as, but not limited to, therapeutic, diagnosticand targeting moieties. Target molecules include, but are not limitedto, polypeptides such as, but not limited to, interferons (includingalpha, beta and gamma), growth hormone, interleukins, enzymes,antibodies (including antibody fragments and monoclonal antibodies),blood factors (including GCSF, erythropoietin, and Factor VIII) andinsulin. In addition, it is intended that the POZ derivatives of thecurrent disclosure be coupled to carbohydrates, oligonucleotides andsmall-molecule therapeutics.

The heterofunctional polyoxazoline derivatives of the presentdisclosure, possessing functional groups that have orthogonalchemistries, are ideally suited for preparation of polyoxazolinederivatives linked to target molecules with distinct functions. Such anapproach is possible due to the presence of two or more functionalgroups on the polyoxazoline derivative that are chemically orthogonal toone another. As a result, target molecules with different linkagechemistries can be incorporated into the polyoxazoline derivative in acontrolled, directed manner. Such an approach is capable of producingtargeted polyoxazoline therapeutic and/or diagnostic conjugates.Exemplary active groups, binding partners on target molecules andlinkages formed there between are disclosed in PCT Application No.PCT/US2008/078159, which is hereby incorporated by reference for suchteaching.

In one embodiment, the target molecule may a therapeutic and/ordiagnostic agent and a targeting agent. The linkages to the targetmolecule may be hydrolytically stable or hydrolytically unstable or acombination thereof. However, in one embodiment, all linkages in thepolyoxazoline derivative itself are hydrolytically stable. For example,an ester linkage to a target molecule would be hydrolytically unstablein vivo, while an amide linkage would be stable for an extended periodof time. The described polyoxazoline derivatives provide a wide range ofoptions in linking target molecules to the described polymer structure.Depending on the number of functional groups present, the number oftarget molecules linked to the polyoxazoline derivative can be variedfrom 1 to at least 100.

EXAMPLES Reagents

Reagents were acquired from EM Science, Oakwood Products, Fluka,Calbiochem, Chevron Phillips Chemicals International, or Aldrich anddistilled before use. Chlorobenzene and oxazolines were distilled fromcalcium hydride. GPC was performed on an Agilent Technologies machinewith an 1100 quaternary pump and RI detector. Two Phenogel™ GPC columns(Phenomenex, 5μ, 500 A° and 1000 A°, 300×7.8 mm) were used in series ina column heater (60° C.). The mobile phase was 100%N,N′-dimethylformamide (DMF) at a flow rate of 1 mL/min. A calibrationcurve was generated with M-PEOZ-OH and H-PEOZ-COOH samples of differentmolecular weights as determined by MALDI (750, 1K, 2K, 5K, 10K, 20K, 30Kand 40K). MALDI-TOF MS was performed with a Bruker, Microflex™ machineusing dithranol as matrix. NMR was performed on a Varian 500 MHzmachine.

Example 1 Preparation of Random-Co-Polymer of with Two Pendent Groups

A solution of monomers comprising 2-(4-pentynyl)-2-oxazoline(PtynOZ—0.274 g, 0.002 mol), T-methyl propionate-2-oxazoline(TMPOZ—0.463 g, 0.002 mol), and 2-ethyl-2-oxazoline (EOZ—1.62 mL, 0.016mol) was prepared in chlorobenzene (10 mL). To this solution was addedmethyl triflate (MeOTf—0.113 mL, 0.001 mol) at room temperature. Afterstirring for 30 minutes, the mixture was heated to 110° C. for 45minutes. The mixture was cooled to 0° C. and then terminated usingpiperidine (0.30 mL, 0.003 mol). After stirring for 2 hours at roomtemperature, the mixture was dripped into diethyl ether, decanted, anddried in vacuo to give 2.4 g of a white powder in quantitative yield.

The product had a calculated Mn of 2400 Da and a Mn (as determined byGPC) of 2440 with a polydispersity index (PDI) of 1.14). The sampleexhibited a high molecular weight shoulder of less than 3%. ¹H NMRspectra showed peaks corresponding to pendent groups (acetylene andmethyl ester) in the proper ratio.

Example 2 Preparation of Block-Co-Sol Mer Via Stepwise Addition ofMonomer and Monomer Mixtures

To a solution of PtynOZ (0.274 g, 0.002 mol, 2 eq) in chlorobenzene (7.5mL) was added MeOTf (0.113 mL, 0.001 mol, 1 eq) at room temperature. Themixture was stirred for 15 minutes and then ethyl oxazoline (EOZ, 0.5mL, 0.005 mol, 5 eq) was added. The mixture was heated to 110° C. andstirred for 17 minutes. To introduce the second block (homopolymer ofEOZ), EOZ (1.01 mL, 0.01 mol, 10 eq) was added and the resulting mixturewas stirred for 10 minutes at 110° C. The third block was introduced bythe addition of a solution of T-methyl propionate oxazoline (TMPOZ,0.463 g, 0.002 mol, 2 eq) and EOZ (0.5 mL, 0.005 mol, 5 eq). Afterstirring for 10 minutes at 110° C., the mixture was cooled to roomtemperature using an ice/water bath and terminated by the addition ofpiperidine (0.4 mL, 0.004 mol). The resulting mixture was allowed tostir overnight, and precipitated by addition to diethyl ether. Thesolution was decanted and the remaining material was dried in vacuo togive 1.8 g of the desired product as a white powder in 67% yield.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaksat 1.12 ppm (m, 3H, CH₃CH₂CO—); 2.30 ppm (m) and 2.41 (s) (total area2H, CH₃CH₂CO—); and 3.46 ppm (m, 4H, —NCH₂CH₂N—). The pentynyl pendantgroup peaks appear at 1.86 ppm (m, 2H, —CH₂CH₂C≡CH), 1.98 ppm (s, 1H,—CH₂CH₂C≡CH), and 2.04 ppm (br s, 1H, —CH₂CH₂C≡CH). The TMP pendentgroup peaks show at 2.60-2.88 ppm (m, 7H, —C(═O)CH(CH₃)CH₂SCH₂CH₂CO₂Me)and 3.67 ppm (s, 3H, CH₂CO₂Me). The ratio of Ptyn, TMP, and EOZ wasdetermined as 2:1.7:20. GPC gave Mn=2510 Da with PDI of 1.14. MALDIprovided Mn=2725 with PDI of 1.04.

Example 3 Synthesis of random H-(Ptyn)₄(EOZ)₂₀-T-CO₂H

Triflic acid (HOTf, 0.177 mL, 0.002 mol) was added into a solution of2-pentynyl-2-oxazoline (PtynOZ, 1.097 g, 0.008 mol, 4 eq) and2-ethyl-2-oxazoline (EOZ, 4.04 mL, 0.04 mol, 20 eq) in chlorobenzene (20mL). After stirring for 5 minutes at room temperature, the mixture washeated to 110° C. for 30 minutes followed by cooling to room temperatureusing an ice/water bath. In a separate flask, the terminating reagentwas prepared by the dropwise addition of methyl 3-mercaptopropionate(0.87 mL, 0.008 mol) into a suspension of sodium hydride (60% in mineraloil, 0.24 g, 0.006 mol) in chlorobenzene (60 mL), at room temperature.This mixture was stirred for 2 hours, before the solution ofH-(Ptyn)₄(EOZ)₂₀ ⁻ in chlorobenzene was slowly added. The resultingmixture was then stirred for 18 hours at room temperature. The organicsolvent was evaporated with a rotary evaporator and the white residuewas dissolved in water and the pH was adjusted to 12.0. After stirringfor 1 hour, the mixture was acidified (pH˜3) and purified byion-exchange chromatography using SP Sepharose FF and DEAE Sepharose FFto give the desired product as a white powder (yield was 2.2 g, 42%).

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaksat 1.13 ppm (m, 3H, CH₃CH₂CO—); 2.32 ppm (m) and 2.41 (s) (total area2H, CH₃CH₂CO—); and 3.47 ppm (m, 4H, —NCH₂CH₂N—). The terminal grouppeaks appear at 2.63 ppm (m, 2H, —SCH₂CH₂CO₂H), 2.74 ppm (m, 2H,—CH₂SCH₂CH₂ CO₂H), and 2.85 ppm (m, 2H, —SCH₂CH₂CO₂H). The pendentpentynyl group peaks appear at 1.85 ppm (m, 2H, —CH₂CH₂C≡CH) and 2.03ppm (br s, 1H, —CH₂CH₂C≡CH). The ratio of Ptyn and EOZ was 4:20. GPCgave Mn=3100 Da and Mp=3140 Da with PDI of 1.05. MALDI provided Mn=2900Da with PDI of 1.03.

Example 4 Synthesis of random H-(Ptyn)₄(EOZ)₂₀-T-NH₂

Triflic acid (HOTf, 0.177 mL, 0.002 mol) was added into a solution of2-pentynyl-2-oxazoline (PtynOZ, 1.097 g, 0.008 mol, 4 eq) and2-ethyl-2-oxazoline (EOZ, 4.04 mL, 0.04 mol, 20 eq) in chlorobenzene (20mL). After stirring for 5 minutes at room temperature, the mixture washeated to 110° C. for 30 minutes followed by cooling to room temperatureusing an ice/water bath. In a separate flask, the terminating reagentwas prepared by the dropwise addition of N-Boc cysteamine (1.01 mL,0.006 mol) into a suspension of sodium hydride (60% in mineral oil, 0.24g, 0.006 mol) in chlorobenzene (60 mL), at room temperature. Thismixture was stirred for 2 hours, before the solution ofH-(Ptyn)₄(PEOZ)₂₀ ⁺ in chlorobenzene was added dropwise. The resultingmixture was stirred for 18 hours at room temperature. Volatiles wereremoved using a rotary evaporator and the residue was dissolved inwater. The pH of the solution was adjusted to 3.0. The resulting aqueoussolution was passed through an Amberlite column and then an ion-exchangecolumn using SP Sepharose FF. The aqueous solution was charged with NaCl(15% w/w) and extracted with dichloromethane. The combined organicphases were dried over anhydrous sodium sulfate, filtered, andconcentrated using a rotary evaporator to provide 4.73 g ofH-(Ptyn)₄(EOZ)₂₀-T-NHBocas a white powder in 87% yield.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaksat 1.12 ppm (m, 3H, CH₃CH₂CO—); 2.28 ppm (m) and 2.41 (s) (total area2H, CH₃CH₂CO—); and 3.47 ppm (m, 4H, —NCH₂CH₂N—). The terminal grouppeaks appear at 1.44 ppm (s, 9H, —NHBoc), 2.63 ppm (m, 2H,—SCH₂CH₂NHBoc), 2.71 ppm (m, 2H, —CH₂SCH₂CH₂NHBoc), and 3.30 ppm (m, 2H,—SCH₂CH₂NHBoc). The pendent group peaks show at 1.84 ppm (m, 2H,—CH₂CH₂C≡CH) and 2.04 ppm (br s, 1H, —CH₂CH₂C≡CH). The ratio of Ptyn andEOZ was determined to be 4:20. GPC gave Mn=3900 Da and Mp=4505 Da withPDI of 1.07.

H-(Ptyn)₄(EOZ)₂₀-T-NHBoc (4.4 g) was dissolved in 3N methanolic HCl andthen stirred for 1 hour at room temperature. Most of volatiles wereremoved using a rotary evaporator and the residue was dissolved in waterand the pH adjusted to ˜12.5. The aqueous solution was charged with NaCl(15% w/w) and extracted with dichloromethane. The combined organicphases were dried over sodium sulfate, filtered, and concentrated usinga rotary evaporator to provide 3.9 g of H-(Ptyn)₄(EOZ)₂₀-T-NH₂as a whitepowder. Deprotection of —NHBoc was confirmed by AKTA prime on SPSepharose FF column media and by ¹H NMR spectra showing thedisappearance of -Boc group peak at 1.44 ppm and the shifting of —CH₂NH₂from 3.30 ppm to 2.92 ppm. In addition, the comparison of integrationshows that the polymer contains four pendant groups. GPC gave Mn=3067 Daand Mp=3927 Da with PDI of 1.16.

Example 5 Synthesis of random H-(NHBoc)₄(EOZ)₂₀-T-CO₂H

Triflic acid (HOTf, 0.177 mL, 0.002 mol) was added into a solution ofT-NHBoc-2-oxazoline (NHBocOZ, 1.097 g, 0.008 mol, 4 eq) and2-ethyl-2-oxazoline (EOZ, 4.04 mL, 0.04 mol, 20 eq) in chlorobenzene (20mL). After stirring for 5 minutes at room temperature, the mixture washeated to 110° C. for 30 minutes followed by cooling to room temperatureusing an ice/water bath. In a separate flask, the terminating reagentwas prepared by the dropwise addition of methyl 3-mercaptopropionate(0.87 mL, 0.008 mol) into a suspension of sodium hydride (60% in mineraloil, 0.24 g, 0.006 mol) in chlorobenzene (60 mL) at room temperature.This mixture was stirred for 2 hours before the solution ofH-(NHBoc)₄(EOZ)₂₀ ⁺ in chlorobenzene was slowly added into it. Theresulting mixture was stirred for 18 hours at room temperature. Theorganic solvent and other volatiles were removed using a rotaryevaporator and the residue was dissolved in water and the pH of thisaqueous solution was adjusted to 12.0. After stirring for 1 hour, themixture was acidified (pH˜3) and purified by ion-exchange chromatographyusing SP Sepharose FF and DEAE Sepharose FF to give the desired productas a white powder (yield was 1.4 g, 22%).

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaksat 1.13 ppm (m, 3H, CH₃CH₂CO—); 2.31 ppm (m) and 2.41 (s) (total area2H, CH₃CH₂CO—); and 3.47 ppm (m, 4H, —NCH₂CH₂N—). The terminal grouppeaks appear at 2.63 ppm (m, 2H, —SCH₂CH₂CO₂H), 2.73 ppm (m, 2H,—CH₂SCH₂CH₂ CO₂H), and 2.84 ppm (m, 2H, —SCH₂CH₂CO₂H). The significantpendent group peaks show at 1.43 ppm (s, 9H, —NHBoc) and 3.28 ppm (m,2H, —CH₂NHBoc). The ratio of NHBoc and EOZ was determined as 4:20. GPCgave Mn=3760 Da and Mp=3550 Da with PDI of 1.09. MALDI provided Mn=3130with PDI of 1.03.

Example 6 Synthesis of random H-(TPA)₄(EOZ)₂₀-T-NHBoc

Triflic acid (HOTf, 0.177 mL, 0.002 mol) was added into a solution ofT-methyl propionate-2-oxazoline (TMPOZ, 1.85 g, 0.008 mol, 4 eq) and2-ethyl-2-oxazoline (EOZ, 4.04 mL, 0.04 mol, 20 eq) in chlorobenzene (20mL). After stirring for 5 minutes at room temperature, the mixture washeated to 110° C. for 30 minutes followed by cooling to room temperatureusing an ice/water bath. In a separate flask, the terminating reagentwas prepared by the dropwise addition of N-Boc cysteamine (1.01 mL,0.006 mol) into a suspension of sodium hydride (60% in mineral oil, 0.24g, 0.006 mol) in chlorobenzene (60 mL) at room temperature. The mixturewas stirred for 2 hours, and the solution of H-(TMP)₄(EOZ)₂₀ ⁺ inchlorobenzene was then slowly added into it. The resulting mixture wasstirred for 18 hours at room temperature. The volatiles were thenremoved using a rotary evaporator and the residue was dissolved in water(pH˜3). The resulting aqueous solution was passed through an Amberlitecolumn and then an ion-exchange column with SP Sepharose FF media. Theaqueous solution was charged with NaCl (15% w/w) and extracted withdichloromethane. The combined organic phases were dried over sodiumsulfate, filtered, and concentrated using a rotary evaporator to provide4.8 g of H-(TMP)₄(EOZ)₂₀-T-NHBoc as a white powder (78% yield).

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaksat 1.12 ppm (m, 3H, CH₃CH₂CO—); 2.30 ppm (m) and 2.41 (s) (total area2H, CH₃CH₂CO—); and 3.46 ppm (m, 4H, —NCH₂CH₂N—). The terminal grouppeaks appear at 1.44 ppm (s, 9H, —NHBoc), 2.61 ppm (m, 2H,—SCH₂CH₂NHBoc), 2.79 ppm (m, 2H, —CH₂SCH₂CH₂NHBoc), and 3.31 ppm (m, 2H,—SCH₂CH₂NHBoc). The pendent group peaks show at 2.60-2.90 ppm (m, 7H,—C(═O)CH(CH₃)CH₂SCH₂CH₂CO₂Me) and 3.67 ppm (s, 3H, CH₂CO₂Me). The ratioof TMP and EOZ was determined as 3.3:20. GPC gave Mn=2340 Da and Mp=2200Da with PDI of 1.08.

H-(TMP)₄(EOZ)₂₀-T-NHBoc (4.4 g) was dissolved in water and the pHadjusted to ˜12.5. After stirring for 1 hour at room temperature,purification was performed by ion-exchange chromatography using DEAESepharose FF. The aqueous solution was charged with NaCl (15% w/w) andextracted with dichloromethane. The combined organic phases were driedover anhydrous sodium sulfate, filtered, and concentrated using a rotaryevaporator to provide 2.0 g of H-(TPA)₄(EOZ)₂₀-T-NHBoc as a white powderin 32% yield. Hydrolysis of —CO₂Me was confirmed by AKTA prime on DEAESepharose FF and by ¹H NMR spectra (disappearance of —CO₂Me group peakat 3.67 ppm).

Example 7 Synthesis of H-[(TPA)₄(EOZ)₂₀][(NHBoc)₄CEOZ)₂₀]-T-Bz

Triflic acid (HOTf, 88.5 μL, 0.001 mol) was added into a solution ofT-methyl propionate-2-oxazoline (TMPOZ, 0.925 g, 0.004 mol, 4 eq) and2-ethyl-2-oxazoline (EOZ, 2.02 mL, 0.02 mol, 20 eq) in chlorobenzene (12mL). After stirring for 5 minutes at room temperature, the mixture washeated to 110° C. for 30 minutes and then a solution ofT-NHBoc-2-oxazoline (NHBocOZ, 1.154 g, 0.004 mol, 4 eq) and2-ethyl-2-oxazoline (EOZ, 2.02 mL, 0.02 mol, 20 eq) in chlorobenzene (12mL) was added. After heating for an additional 30 minutes, the mixturewas cooled to room temperature using an ice/water bath. The terminatingreagent was prepared in a separate flask by the slow addition of benzylmercaptan (0.35 mL, 0.003 mol) into a suspension of sodium hydride (60%in mineral oil, 0.08 g, 0.002 mol) in chlorobenzene (10 mL) at roomtemperature. After the mixture was stirred for 2 hours, the solution ofliving polymer species in chlorobenzene was added dropwise to thetermination mixture. The resulting mixture was stirred for 18 hours atroom temperature and then precipitated by addition to diethyl ether. Theprecipitated solution was filtered, and dried to give 4.8 g of polymerhaving methyl ester and —NHBoc as the pendent groups. The polymer wasdissolved in water, passed through an Amberlite column and then anion-exchange column with SP Sepharose FF packing. The resulting aqueoussolution was charged with NaCl (15% w/w) and extracted withdichloromethane. The combined organic phases were dried over sodiumsulfate, filtered, and concentrated to provide 3.72 g ofH-[(TMP)₄(EOZ)₂₀][(NHBoc)₄(EOZ)₂₀]-T-Bz as a white powder in 78% yield.

¹-H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbonepeaks at 1.12 ppm (m, 3H, CH₃CH₂CO—); 2.30 ppm (m) and 2.41 (s) (totalarea 2H, CH₃CH₂CO—); and 3.47 ppm (m, 4H, —NCH₂CH₂N—). The terminalgroup peaks appear at 2.56 ppm (m, 2H, —CH₂SCH₂Ar), 3.74 ppm (s, 2H,—SCH₂Ar), 7.27 ppm (m, 1H, —Ar), 7.34 ppm (m, 5H, —Ar). The TMP pendentgroup peaks show at 2.60-2.88 ppm (m, 7H, —C(═O)CH(CH₃)CH₂SCH₂CH₂CO₂Me)and 3.67 ppm (s, 3H, CH₂CO₂Me). The —NHBoc pendent group peaks show at1.42 ppm (s, 9H, —NHBoc) and 3.28 ppm (m, 2H, —CH₂NHBoc). The ratio ofTMP, NHBoc, and EOZ was determined as 3.5:2:40. GPC gave Mn=4050 Da andMp=4690 Da with PDI of 1.16.

H—RTMP)₄(EOZ)₂₀][(NHBoc)₄(EOZ)₂₀]-T-Bz(0.8 g) was dissolved in water andthe pH was adjusted to ˜13.0 using 0.5 M NaOH solution. After stirringfor 1 hour, the mixture was extracted with dichloromethane and thecombined organic phases were dried over sodium sulfate, filtered, andconcentrated using a rotary evaporator. The resulting solution wasprecipitated by addition to diethyl ether. The diethyl ether solutionwas decanted and the remaining white powdery material was dried in vacuoto give the desired product, H-[(TPA)₄(EOZ)₂₀][(NHBoc)₄(EOZ)₂₀]-T-Bz ina quantitative yield. The hydrolysis was confirmed by ion exchangechromatography using a DEAE Sepharose FF. GFC and ¹H NMR showed thehydrolysis of the —CO₂Me group.

Example 8 Synthesis of M-(PPtyn)₂(PEOZ)₁₈-T-CO₂H

Methyl triflate (MeOTf, 0.556 mL, 0.005 mol) was added into a solutionof 2-pentynyl-2-oxazoline (PtynOZ, 1.37 g, 0.01 mol, 2 eq) inchlorobenzene (20 mL). After stirring for 10 minutes at roomtemperature, 2-ethyl-2-oxazoline (9.09 mL, 0.09 mol, 18 eq) was addedand the mixture was heated to 110° C. for 30 minutes followed by coolingto 0° C. To obtain a terminating reagent, methyl 3-mercaptopropionate(2.17 mL, 0.02 mol) was added dropwise into a suspension of potassiumtert-butoxide (1.12 g, 0.01 mol) in chlorobenzene (10 mL) at 0° C. Afterthe mixture was stirred for 2 hours in the cold, the solution ofM-(PPtyn)(PEOZ)⁺ in chlorobenzene was added dropwise. The mixture wasstirred in the cold for 4 hours and then stirred for 18 hours at roomtemperature. Water (100 mL) was added and the mixture was acidified(pH˜3) by the addition of 5% aqueous HCl solution. Most of volatilesincluding chlorobenzene were removed using rotary evaporation. Theresulting aqueous solution was treated with NaOH (1.0 g, 0.025 mol).After stirring for 1 hour, the mixture was acidified with 5% aqueous HClsolution and then extracted with dichloromethane. The combined organicphases were dried over sodium sulfate, filtered, concentrated, andprecipitated by addition to ether. The ether was decanted and theresidue was dried under vacuum. GFC showed that the crude productcontains hydroxyl-terminated polymer (20%) and the desiredacid-terminated polymer (80%). Further purification was performed byion-exchange chromatography using DEAE Sepharose FF to give 5.6 g of theproduct in 61% yield. NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) shows peaksfor pendant pentynyl group at 1.85 ppm (m, 2H, —CH₂CH₂C≡CH); and 1.98ppm (m, 1H, —CH₂CH₂C≡CH). GPC and GFC show a single main peak, with Mn1830 Da and PD of 1.10.

Example 9 Synthesis of {M-(PPtyn)₂(PEOZ)₁₈}₂-Lys-CO₂H

A solution of M-(PPtyn)₂(PEOZ)₁₈-T-CO₂H (Mn=1830 Da, 2.0 g, 1.09 mmol)and 1-HOBT (0.351 g, 2.60 mmol) in acetonitrile (40 mL) was concentratedby rotary evaporation to dry azeotropically. The residue was dissolvedin dry CH₂Cl₂ (20 mL) and then DCC (0.322 g, 1.56 mmol) was added. Afterthe mixture was stirred for 3 hours at room temperature, L-lysine ethylester dihydrochloride (0.129 g, 0.520 mmol) and DMAP (0.318 g, 2.60mmol) were added. After stirring for 18 hours at room temperature, themixture was added to ether (150 mL) to give a white precipitate. Thecompound was filtered and dried under vacuum to give{M-(PPtyn)₂(PEOZ)₁₈}₂-Lys-ethyl ester as a white powder. GPC and GFCshowed the mixture of main product (98%, Mn=4540 Da, PD=1.04) and excessof acid polymer (2. The crude product was passed through a DEAESepharose FF column to remove the excess amount of acid polymer. Theresulting aqueous solution was treated with NaOH (0.104 g , 2.60 mmol)for 1 hour. The mixture was acidified with 5% aqueous HCl solution andthen extracted with dichloromethane. The combined organic phases weredried over sodium sulfate, filtered, concentrated, and precipitated byaddition to ether. The ether was decanted and the residue was driedunder vacuum to give 2.0 g of a white powder in 92% yield. ¹H NMR showedthe completion of hydrolysis according to the disappearance of ethylgroup peaks and peaks for lysine core at 1.42 ppm (br s, 2H,—C(═O)NHCH₂CH₂CH₂—); 1.54 ppm (br s, 2H, —C(═O)NHCH₂CH₂—); 1.85 ppm (m,2H, —CH₂CH(CO₂H)NH—); and 4.52 ppm (m, 1H, —CH₂CH(CO₂H)NH—). MALDIprovided Mn=4240 Da with PD of 1.01.

Example 10 Synthesis of {M-(PPtyn)(PEOZ)}₂Lys-NHS

N-hydroxysuccinimide (0.0235 g, 0.204 mmol) and DCC (0.0421 g, 0.204mmol) were added into a solution of {M-(PPtyn)₂(PEOZ)₁₈}₂-Lys-CO₂H (Mn4200 Da, 0.832 g, 0.198 mmol) in dichloromethane (4 mL) at 0° C. Afterstirring for 2 hours in the cold, the mixture was warmed to roomtemperature and stirred overnight. The white precipitate was removed byfiltration and the solution was added to diethyl ether to give a whitepowder. The powder was collected by filtration and dried under vacuum(0.8 g, 88% yield). The attachment of maleimide was shown by ¹H NMRspectrum that shows the succinimidyl protons at 2.86 ppm (s, 2H) alongwith the usual backbone peaks. GPC showed 97% of the desired product(Mn=4550 Da, PD=1.04) and 3% of acid polymer.

To confirm product identity, the product (Mn 4550 Da, 0.103 g, 0.023mmol) was treated with phenylethylamine (0.009 mL, 0.068 mmol) andtriethylamine (0.009 mL, 0.068 mmol) in dichloromethane (3 mL). Afterstirring overnight, the mixture was filtered and added to diethyl ether.The white powder was isolated by filtration and dried under vacuum toprovide the product in quantitative yield. According to GFC, theconversion yield was 99.7%.

Example 11 Synthesis of POZ-4[{-(PPtyn),(PEOZ)₁₈}₂-Lys]₂-Lys-ethyl ester

A solution of M-{(PPtyn)₂(PEOZ)₁₈}₂Lys-T- CO₂H (Mn=4200 Da, 0.415 g,0.0988 mmol) and 1-HOBT (0.0318 g, 0.235 mmol) in acetonitrile (15 mL)was concentrated using rotary evaporation. The residue was dissolved indry CH₂Cl₂ (3 mL) and then DCC (0.0291 g, 0.141 mmol) was added. Afterthe mixture was stirred for 3 hours at room temperature, L-lysine ethylester dihydrochloride (0.0116 g, 0.047 mmol) and DMAP (0.0287 g, 0.235mmol) were added. After stirring for 18 hours at room temperature, themixture was filtered and added to ether (40 mL) to give a whiteprecipitate. The precipitate was isolated by filtration and dried undervacuum to give the desired four-armed[{M-(PPtyn)₂(PEOZ)₁₈}₂-Lys]₂-Lys-ethyl ester as a white powder inquantitative yield. ¹-H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed thepeak for Lys-Lys-Lys core molecule at 1.28 ppm (t, 3H, CH₃CH₂O—); 1.42ppm (m, 6H, —C(═O)NHCH₂CH₂CH₂—); 1.54 ppm (m, 6H, —C(═O)NHCH₂CH₂—); 1.85ppm (m, 6H, —CH₂CH(CO₂H)NH—); 4.14 ppm (br s, 2H, —CO₂CH₂CH₃) and 4.45ppm (m, 3H, —CH₂CH(CO₂H)NH—) and peaks for pendant pentynyl group at1.85 ppm (m, 2H, —CH₂CH₂O≡CH); and 2.02 ppm (m, 1H, —CH₂CH₂C≡CH). GPCand GFC showed a mixture of main product (96%, Mn=7970 Da, PD=1.06) andexcess of acid polymer (4%).

Example 12 Conjugation of Glucosamine and Zidovudine ontoH-(Ptyn)₄(EOZ)₂₀-T-CO₂H

After drying azeotropically with acetonitrile, H-(Ptyn)₄(EOZ)₂₀-T-CO₂H(0.18g, 0.0619 mmol, Mn 2100 Da by MALDI) was dissolved indichloromethane (2 mL). NHS (0.0071 g, 0.0619 mmol) and DCC (0.0128 g,0.0619 mmol) were added at room temperature. After stirring overnight atroom temperature, the mixture was filtered and precipitated by additionto diethyl ether. Diethyl ether solution was decanted and the residuewas dried in vacuo to give the desired N-hydroxysuccinimide ester; i.e.H-(Ptyn)₄(EOZ)₂₀-T-SPA as a white powder in a quantitative yield. GFCshows purity and attachment of NHS was proved by ¹H NMR showing thesuccinimidyl protons at 2.86 ppm (s, 4H).

D(+)-glucosamine hydrochloride (0.137 g, 0.0663 mmol) was dissolved in 2mL of 0.1 N boric acid solution followed by the adjustment of pH to 8.5using 0.1 N NaOH solution. H-(Ptyn)₄(EOZ)₂₀-T-SPA (0.19 g, 0.0663 mmol,Mn 3000 Da) was added as a solid. The mixture was stirred for 3 hours atroom temperature while maintaining pH at 8.5 using 0.1 N NaOH.

The mixture was acidified (pH˜3) and extracted with dichloromethane. Thecombined organic phases were dried over anhydrous Na₂SO₄, filtered,concentrated, and dried in vacuo to give 0.14 g of pale yellow powder.The conjugation of glucosamine was confirmed by ¹H NMR (Varian, 500 MHz,10 mg/mL DMSO-d6) that shows the 2-position proton on glucoseamide at4.92 ppm (s, 1H). Ion exchange chromatography showed a substitutionyield of 83%.

H-(Ptyn)₄(EOZ)₂₀-T-Gluco (0.05g, 0.0156 mmol, Mn 3200 Da) and theantiviral nucleoside zidovudine (AZT, 0.0167g, 0.0625 mmol, 4 eq) weredissolved in water (2 mL). Sodium ascorbate (0.0012 g, 0.00625 mmol) andCuSO₄.5H₂O (0.0008 g, 0.00313 mmol) were added at room temperature.After stirring for 18 hours, water was removed using a rotaryevaporator. The residue was dissolved in a 1:1 mixture of MeOH and CHCl₃and then precipitated by addition to diethyl ether. Diethyl ethersolution was decanted and the residue was dried in vacuo to give thedesired product as a white powder in a quantitative yield. The ‘click’coupling of the azide group on zidovudine to each acetylene pendant onthe polymer chain was verified by NMR. ¹H NMR (Varian, 500 MHz, 10 mg/mLDMSO-d6) shows that the polymer chain contained an average of units ofthymidines with the thymidyl proton peaks at 11.3 ppm (br s, 1H, —OH),8.05 ppm (s, 1H, triazole), 7.81 ppm (s, 1H), 6.41 ppm (t, 1H), 5.31 ppm(m, 1H), 5.26 ppm (m, 1H), 4.18 ppm (br s, 1H), and 1.80 (s, 3H). The2-position proton on glucoseamide shows at 4.92 ppm (s, 1H). GPC gaveMn=4700 Da and Mp=4830 Da with PDI of 1.07.

Example 13 Conjugation of Irinotecan to POZ

Irinotecan.HCl.3H₂O (0.200 g, 0.295 mmol) was dissolved in acetonitrile(15 mL) and dried by azeotropic distillation. The residue was dissolvedin dichloromethane (6 mL) and 6-azidohexanoic acid (0.0928 g, 0.591mmol) was added. After the addition of dimethyaminopyridine (DMAP)(0.0722 g, 0.591 mmol) and dicyclohexylcarbodiimide (DCC) (0.122 g,0.591 mmol), the resulting mixture was allowed to stir overnight at roomtemperature. The mixture was precipitated by addition to diethyl ether.The ether solution was decanted and the remaining precipitate was driedto give 0.144 g of the desired product as a pale yellow powder (67%yield). ¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) δ 0.98 (t, 3H, C-9H),1.41 (m, 2H, C-3H), 1.41 (m, 3H, C-18H), 1.41 (m, 2H, bipiperidyl H),1.58 (m, 2H, C-4H), 1.58 (2H, bipiperidyl H), 1.70 (m, 2H, C-2H), 1.93(m, 4H, bipiperidyl H), 2.16 (m, 1H, C-1H), 2.28 (m, 1H, C-1H), 2.32 (m,1H, bipiperidyl H), 2.50 (m, 2H, C-5H), 2.50 (m, 2H, bipiperidyl H),2.75 (m, 2H, C-17H), 2.94 (m, 1H, bipiperidyl C-3′H), 3.17 (m, 3H,bipiperidyl H), 3.25 (t, 2H, C-1H, —CH₂N₃), 3.55 (br s, 2H, bipiperidylC-1′H and C-5′H), 4.56 (m, 2H, bipiperidyl C-1′H and C-5′H), 5.25 (s,2H, C-14H), 5.41 (d, 1H, C-11H), 5.68 (d, 1H, C-11H), 7.19 (s, 1H,C-27H), 7.60 (d, 1H, C-22H), 7.87 (s, 1H, C-20H), 8.23 (d, 1H, C-23H).

Conjugation of random H-(Ptyn)₄(EOZ)₂₀-T-NH₂ and Irinotecan AzidoHexanoate

H-(Ptyn)₄(EOZ)₂₀-T-NH₂ (0.05 g, 0.0163 mmol, 1 eq., Mn 3070 Da), from aprevious example, and irinotecan azido hexanoate (0.0473 g, 0.0652 mmol,4 eq) were dissolved in water (2 mL) Sodium ascorbate (0.0013 g, 0.00652mmol, 0.4 eq) and CuSO₄.5H₂O (0.0008 g, 0.00326 mmol, 0.2 eq) were addedat room temperature. After stirring for 22 hours, water was removedusing a rotary evaporator. The residue was dissolved in dichloromethaneand then precipitated by addition to diethyl ether. Diethyl ethersolution was decanted and the residue was dried in vacuo to give 0.7g ofthe desired product as an off-white powder (72% yield). The ‘click’coupling of the azide group (on irinotecan azido hexanoate) to theacetylene pendants (on POZ) was verified by NMR. ¹H NMR (Varian, 500MHz, 10 mg/mL CDCl₃) shows that the polymer chain contained an averageof 4 units of irinotecans with the aromatic proton peaks of irinotecanat 7.14 ppm (t, 1H), 7.57 ppm (br s, 1H), and 7.85 ppm (br s, 1H), 8.16ppm (br s, 1H), and new signals at 4.27 ppm (br s, 2H, adjacent totriazole —CH₂NR) and 7.40 ppm (br s, 1H, triazole). GPC gave Mn=4160 Daand Mp=4900 Da with PDI of 1.19.

1. A heterofunctional polyoxazoline derivative of the general structure:R₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂)]_(m)}_(a)-Zwherein: R₁ is an initiating group; R₂ is independently selected foreach repeating unit from an unsubstituted or substituted alkyl, anunsubstituted or substituted alkenyl, an unsubstituted or substitutedaralkyl or an unsubstituted or substituted heterocyclylalkyl group; X isa pendent moiety containing a first functional group; Y is a pendentmoiety containing a second functional group; Z is a terminatingnucleophile, wherein Z is inert or wherein Z contains a third functionalgroup; a is ran which indicates a random copolymer or block whichindicates a block copolymer; o and m are each an integer independentlyselected from 1-50; n is an integer selected from 0-1000; and wherein atleast two functional groups are chemically orthogonal to one another. 2.The heterofunctional polyoxazoline derivative of claim 1 wherein R₁ is ahydrogen, substituted or unsubstituted alkyl or substituted orunsubstituted aralkyl group.
 3. The heterofunctional polyoxazolinederivative of claim 1 wherein the first and second functional groups areeach independently an alkyne, an amine, an oxyamine, an aldehyde, aketone, an acetal, a ketal, an ester, a carboxylic acid, an activatedcarboxylic acid, an active carbonate, a chloroformate, an alcohol, anazide, a vinyl sulfone, a maleimide or orthopyridyl disulfide and thethird functional group, when present, is an alkyne, an amine, anoxyamine, an aldehyde, a ketone, an acetal, a ketal, an ester, acarboxylic acid, an activated carboxylic acid, an active carbonate, achloroformate, an alcohol, an azide, a vinyl sulfone, a maleimide ororthopyridyl disulfide.
 4. The heterofunctional polyoxazoline derivativeof claim 3 wherein the carboxylic acid is a negatively charged speciesand the amine is a positively charged species.
 5. The heterofunctionalpolyoxazoline derivative of claim 1 wherein Z contains the thirdfunctional group.
 6. The heterofunctional polyoxazoline derivative ofclaim 5 wherein the first and second functional groups are eachchemically orthogonal to the third functional group.
 7. Theheterofunctional polyoxazoline derivative of claim 1 wherein Z is—S—U—W, wherein U is a linking group and W is an alkyne, an amine, anoxyamine, an aldehyde, a ketone, an acetal, a ketal, an ester, acarboxylic acid, an activated carboxylic acid, an active carbonate, achloroformate, an alcohol, an azide, a vinyl sulfone, a maleimide ororthopyridyl disulfide.
 8. The heterofunctional polyoxazoline derivativeof claim 7 wherein W is —CO₂H or —NH₂.
 9. The heterofunctionalpolyoxazoline derivative of claim 5 wherein the first and secondfunctional groups are each chemically orthogonal to the third functionalgroup or wherein at least one of the first and second functional groupsis chemically orthogonal to the third functional group.
 10. Theheterofunctional polyoxazoline derivative of claim 1 wherein Z is inert.11. The heterofunctional polyoxazoline derivative of claim 10 wherein Zis —S-T-V, wherein V is an inert group.
 12. The heterofunctionalpolyoxazoline derivative of claim 11 wherein V is —CO₂CH₃ or —C₆H₅. 13.The heterofunctional polyoxazoline derivative of claim 9 wherein thefirst and second functional groups are chemically orthogonal.
 14. Theheterofunctional polyoxazoline derivative of claim 1 linked to at leastone target molecule to form a target molecule-POZ conjugate.
 15. Amulti-armed heterofunctional polyoxazoline derivative of the generalstructure:{R₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂)]_(m)}_(a)—K_(k)—}_(d)—R-Q_(q)-Zwherein: R₁ is an initiating group; R₂ is independently selected foreach polyoxazoline chain from a substituted or unsubstituted alkyl, asubstituted or unsubstituted alkenyl, a substituted or unsubstitutedaralkyl or a substituted or unsubstituted heterocyclylalkyl group; X isa pendent moiety bearing a first functional group; Y is a pendent moietybearing a second functional group; K is a linking moiety linking eachpolyoxazoline chain to a branching moiety R; Q is a linking moietylinking the branching moiety R to Z; R is a branching moiety capable offorming linkages with Z, either directly or through linking group Q, andwith each polyoxazoline chain, either directly or through the linkinggroup K; Z is a moiety containing a third functional group or an inertgroup; a independently selected for each polyoxazoline chain from ranwhich indicates a random copolymer or block which indicates a blockcopolymer; d is an integer selected from 2-8; k is an integerindependently selected for each polyoxazoline chain from one or zero; qis an integer selected from one or zero; o is an integer independentlyselected for each polyoxazoline chain from 1-50; m is an integerindependently selected for each polyoxazoline chain from 0-50; n is aninteger selected from 0-1000; and wherein at least two functional groupsare chemically orthogonal to one another.
 16. The multi-armed,heterofunctional polyoxazoline derivative of claim 15 wherein Z containsthe third functional group, d is 2 and the first and second functionalgroups are chemically orthogonal to one another and having the generalstructure:{R₁—{[N(COX)CH₂CH₂]_(o)—[N(COR₂)CH₂—CH₂)]_(n)—[N(COY)CH₂—CH₂)]_(m)}_(a)—K_(k)—}₂—R-Q_(q)-Z.17. The multi-armed, heterofunctional polyoxazoline derivative of claim16 wherein R₁ is independently selected for each polyoxazoline chainfrom a hydrogen, substituted or unsubstituted alkyl or substituted orunsubstituted aralkyl group.
 18. The multi-armed, heterofunctionalpolyoxazoline derivative of claim 16 wherein the first and secondfunctional groups are each independently selected for each polyoxazolinechain from an alkyne, an amine, an oxyamine, an aldehyde, a ketone, anacetal, a ketal, an ester, a carboxylic acid, an activated carboxylicacid, an active carbonate, a chloroformate, an alcohol, an azide, avinyl sulfone, a maleimide or orthopyridyl disulfide and the thirdfunctional group is an alkyne, an amine, an oxyamine, an aldehyde, aketone, an acetal, a ketal, an ester, a carboxylic acid, an activatedcarboxylic acid, an active carbonate, a chloroformate, an alcohol, anazide, a vinyl sulfone, a maleimide or orthopyridyl disulfide.
 19. Themulti-armed, heterofunctional polyoxazoline derivative of claim 18wherein the first, second and third functional groups are chemicallyorthogonal to one another or wherein the third functional group ischemically orthogonal to at least one of the first or second functionalgroups.
 20. The multi-armed, heterofunctional polyoxazoline derivativeof claim 19 wherein the carboxylic acid is a negatively charged speciesand the amine is a positively charged species.
 21. The multi-armed,heterofunctional polyoxazoline derivative of claim 16 wherein Q is asubstituted or unsubstituted alkylene.
 22. The multi-armed,heterofunctional polyoxazoline derivative of claim 16 wherein K isindependently selected for each polyoxazoline chain from —(CH₂)_(p)O—,—(CH₂)_(p)—CO—, —S—(CH₂)_(p)CONH—, —S—(CH₂)CO₃—(CH₂)_(p)—NHCSO—,—(CH₂)—NHCO₂—, —NH—(CH₂) or —NHCO₂—, where p is an integer from 0-10.23. The multi-armed, heterofunctional polyoxazoline derivative of claim16 wherein the K group is the same for each polyoxazoline chain ordifferent for each polyoxazoline chain.
 24. The multi-armed,heterofunctional polyoxazoline derivative of claim 16 wherein R is anitrogen, an aryl group, or —CR₃—, where R₃ is hydrogen or a substitutedor unsubstituted alkyl, a substituted or unsubstituted alkenyl, or asubstituted or unsubstituted aralkyl group.
 25. The multi-armed,heterofunctional polyoxazoline derivative of claim 16 wherein R is—NH—CH—(CH₂)₃—CH₂—NH—.
 26. The multi-armed, heterofunctionalpolyoxazoline derivative of claim 16 linked to at least one targetmolecule to form a target molecule-POZ conjugate.
 27. The multi-armed,heterofunctional polyoxazoline derivative of claim 15 wherein Z containsthe third functional group, d is 2, k is 1, K is —S—(CH₂)_(p)—C—, wherep is an integer from 1-10, for each polyoxazoline chain, q is 0, R isNH—CH—(CH₂)₃—CH₂—NH and the first and second functional groups arechemically orthogonal to one another and having the general structure:


28. The multi-armed, heterofunctional polyoxazoline derivative of claim27 wherein R₁ is independently selected for each polyoxazoline chainfrom a hydrogen, substituted or unsubstituted alkyl or substituted orunsubstituted aralkyl group.
 29. The multi-armed, heterofunctionalpolyoxazoline derivative of claim 27 wherein the first and secondfunctional groups are each independently selected for each polyoxazolinechain from an alkyne, an amine, an oxyamine, an aldehyde, a ketone, anacetal, a ketal, an ester, a carboxylic acid, an activated carboxylicacid, an active carbonate, a chloroformate, an alcohol, an azide, avinyl sulfone, a maleimide or orthopyridyl disulfide and Z is CO₂H. 30.The multi-armed, heterofunctional polyoxazoline derivative of claim 29wherein the first and second functional groups are chemically orthogonalto the third functional group or wherein at least one of the first orsecond functional groups is chemically orthogonal to the thirdfunctional group.
 31. The multi-armed, heterofunctional polyoxazolinederivative of claim 29 wherein the carboxylic acid is a negativelycharged species and wherein the amine is a positively charged species.32. The multi-armed, heterofunctional polyoxazoline derivative of claim15 having the structure:

wherein the first functional group is independently an alkyne, an amine,an oxyamine, an aldehyde, a ketone, an acetal, a ketal, an ester, anactive carbonate, a chloroformate, an alcohol, an azide, a vinylsulfone, a maleimide or orthopyridyl disulfide.
 33. The multi-armed,heterofunctional polyoxazoline derivative of claim 32 linked to at leastone target molecule to form a target molecule-POZ conjugate.
 34. Themulti-armed, heterofunctional polyoxazoline derivative of claim 15wherein Z contains the third functional group, d is 4, k is 1 and K is—S—(CH₂)₂—CO—NH for each polyoxazoline chain, q and m are 0, R is—(CH₂)₄—CH—CO—NH—(CH₂)₄—CH—NH—CO—CH—(CH₂)₄— and the first and thirdfunctional groups are chemically orthogonal to one another having thegeneral structure:


35. The multi-armed, heterofunctional polyoxazoline derivative of claim34 wherein R₁ is independently selected for each polyoxazoline chainfrom a hydrogen, substituted or unsubstituted alkyl or substituted orunsubstituted aralkyl group.
 36. The multi-armed, heterofunctionalpolyoxazoline derivative of claim 34 wherein Z is CO₂H and the firstfunctional group is an alkyne, an amine, an oxyamine, an aldehyde, aketone, an acetal, a ketal, an ester, an alcohol, an azide, a vinylsulfone, a maleimide or orthopyridyl disulfide or Z is NH₂ and the firstfunctional group is an alkyne, an oxyamine, an aldehyde, a ketone, anacetal, a ketal, an ester, a carboxylic acid, an activated carboxylicacid, an active carbonate, a chloroformate, an alcohol, an azide, avinyl sulfone, a maleimide or orthopyridyl disulfide.
 37. Themulti-armed, heterofunctional polyoxazoline derivative of claim 36wherein the carboxylic acid is a negatively charged species and whereinthe amine is a positively charged species.
 38. The multi-armed,heterofunctional polyoxazoline derivative of claim 34 linked to at leastone target molecule to form a target molecule-POZ conjugate.
 39. Themulti-armed, heterofunctional polyoxazoline derivative of claim 15wherein the at least one target molecule is linked to the polyoxazolinederivative through at least one of the first, second or third functionalgroups.
 40. The multi-armed, heterofunctional polyoxazoline derivativeof claim 39 wherein the at least one target molecule is a therapeuticmoiety, a diagnostic moiety or a targeting moiety and the targetmolecule-POZ conjugate contains a therapeutic moiety and a targetingmoiety, a diagnostic moiety and a targeting moiety or a diagnosticmoiety, a therapeutic moiety and a targeting moiety.
 41. Themulti-armed, heterofunctional polyoxazoline derivative of claim 26wherein the at least one target molecule is linked to the polyoxazolinederivative through at least one of the first, second or third functionalgroups.
 42. The multi-armed, heterofunctional polyoxazoline derivativeof claim 41 wherein the at least one target molecule is a therapeuticmoiety, a diagnostic moiety or a targeting moiety and the targetmolecule-POZ conjugate contains a therapeutic moiety and a targetingmoiety, a diagnostic moiety and a targeting moiety or a diagnosticmoiety, a therapeutic moiety and a targeting moiety.
 43. Themulti-armed, heterofunctional polyoxazoline derivative of claim 33wherein the at least one target molecule is linked to the polyoxazolinederivative through at least one of the first functional group or theCO₂H group.
 44. The multi-armed, heterofunctional polyoxazolinederivative of claim 43 wherein the at least one target molecule is atherapeutic moiety, a diagnostic moiety or a targeting moiety and thetarget molecule-POZ conjugate contains a therapeutic moiety and atargeting moiety, a diagnostic moiety and a targeting moiety or adiagnostic moiety, a therapeutic moiety and a targeting moiety.
 45. Themulti-armed, heterofunctional polyoxazoline derivative of claim 38wherein the at least one target molecule is linked to the polyoxazolinederivative through at least one of the first or third functional groups.46. The multi-armed, heterofunctional polyoxazoline derivative of claim45 wherein the at least one target molecule is a therapeutic moiety, adiagnostic moiety or a targeting moiety and the target molecule-POZconjugate contains a therapeutic moiety and a targeting moiety, adiagnostic moiety and a targeting moiety or a diagnostic moiety, atherapeutic moiety and a targeting moiety.
 47. The multi-armed,heterofunctional polyoxazoline derivative of claim 27 linked to at leastone target molecule to form a target molecule-POZ conjugate.
 48. Themulti-armed, heterofunctional polyoxazoline derivative of claim 47wherein the at least one target molecule is linked to the polyoxazolinederivative through at least one of the first, second or third functionalgroups.
 49. The multi-armed, heterofunctional polyoxazoline derivativeof claim 48 wherein the at least one target molecule is a therapeuticmoiety, a diagnostic moiety or a targeting moiety and the targetmolecule-POZ conjugate contains a therapeutic moiety and a targetingmoiety, a diagnostic moiety and a targeting moiety or a diagnosticmoiety, a therapeutic moiety and a targeting moiety.